Revealing the Hidden Depths of Proteomes and Phosphoproteomes with a Tickle
Dr. Duncan Smith Paterson Institute for Cancer Research Christie Hospital Manchester, UK

The study of the protein complement of an organism or cell system is invaluable to the modern biochemist. It facilitates the understanding of basic cell biology of organisms in health and disease. Many 'proteomic' studies rely on wholesale proteolytic digestion of a given proteome, followed by analysis using liquid chromatography and mass spectrometry (LCMS). The success of such approaches is highly dependent upon obtaining robust, reproducible and efficient digestion. Many protein subsets are relatively underrepresented in such datasets due to their resilience to proteolytic digestion. The plasma membrane bound proteins are just such a class of proteins that are resilient to digestion, largely due to their hydrophobic nature. This class of proteins are incredibly important because they are components of signalling on which every cell is utterly dependent (and they are very attractive drug targets). Here we present compelling evidence that microwave assisted digestion procedures facilitate the analysis of plasma membrane proteomes to a much greater penetrance than previously obtained with other methods.

Phosphorylation is a key post-translational modification that plays a central role in many cellular activities. The study of the phosphoproteome has massive appeal as the function of up to a third of all proteins expressed are thought to be modulated by phosphorylation at some time in the proteins life cycle. However, the study of phosphopeptides by LCMS represents a considerable challenge and has thus far been the major limiting factor in the success of phosphoproteomics. Here we present a novel methodology based on microwave-assisted dephosphorylation followed by diagonal chromatography that has been successfully applied to the study of the phosphoproteome.